Structure/Function Analysis of Phagocyte Proteins

性状（申请人提供）：吞噬细胞呼吸爆发氧化酶 产生超氧化物自由基在宿主防御中起着核心作用， 炎症反应。活性氧化酶复合物的组装需要 膜和胞质蛋白的参与，并受 小GTP一种吞噬细胞特异性b型黄细胞色素异二聚体，位于 在血浆和中性粒细胞中，特异性颗粒膜是病灶 点氧化酶组装，并含有黄素和血红素氧化还原中心 用于将电子从NADPH转移到分子氧。中的遗传缺陷 氧化酶蛋白，包括两个黄细胞色素亚基，导致慢性 肉芽肿性疾病（CGD），一种以呼吸道疾病缺失为特征的综合征， 爆裂、复发性感染和慢性肉芽肿。的结构和 各种氧化酶亚基和组装之间的功能关系 活性NADPH氧化酶复合物的活性仍不完全清楚。这 低电位黄细胞色素是由gp 91 phox，91-kDa的 由X连锁基因编码的糖蛋白，是 X-连接的CGD，和p221-phOx，一种衍生自 常染色体CGD基因座拟议的研究采取遗传学方法， 研究黄细胞色素氧化酶B的结构和功能， 其作为组装活性NADPH氧化酶复合物的焦点的作用。的 该项目有3个具体目标，利用了GP 91 phox 通过基因靶向开发的缺陷型吞噬细胞细胞系以及 我们已经开发了异源细胞系统，用于表达功能性 重组氧化酶亚基。首先，gp 91 phox和 p22 phox在黄细胞色素B生物合成和功能中的作用将用 用于表达未组装的亚基的异源细胞，所述未组装的亚基在其它情况下是 在吞噬细胞中不稳定。第二，鉴定gp 91 phox和p22中的结构域 在氧化酶活性组装和调节中起作用的phox将 追求，使用强调定点诱变的策略， 候选亲水区域，随后表达和分析功能 在完整的细胞中。第三，对黄细胞色素B重要的功能域 将研究吞噬过程中的运输和NADPH氧化酶组装 在异源和吞噬细胞系中使用类似的方法。这些 研究将进一步深入了解超氧化物生成系统， 吞噬细胞，这可能导致新的方法在调节超氧化物 在炎症反应和宿主防御中的形成。

DESCRIPTION (provided by applicant): The phagocyte respiratory burst oxidase that generates the superoxide radical plays a central role in host defense and the inflammatory response. Assembly of the active oxidase complex requires the participation of both membrane and cytosolic proteins, and is regulated by small GTPases. A phagocyte-specific b-type flavocytochrome heterodimer, located in the plasma and, in neutrophils, specific granule membranes, is the focal point for oxidase assembly, and contains both the flavin and heme redox centers for transfer of electrons from NADPH to molecular oxygen. Genetic defects in oxidase proteins, including the two flavocytochrome subunits, result in chronic granulomatous disease (CGD), a syndrome characterized by an absent respiratory burst, recurrent infections, and chronic granulomas. The structural and functional relationships between the various oxidase subunits and the assembly of the active NADPH oxidase complex remain incompletely understood. This low-potential flavocytochrome is a heterodimer comprised of gp91 phox, a 91-kDa glycoprotein encoded by an X-linked gene that is the site of mutations in X-linked CGD, and p221-phOx, a non-glycosylated peptide derived from an autosomal CGD locus. The proposed studies take a genetic approach to investigating the structure and function of the oxidase flavocytochrome b and its role as a focal point for assembly of the active NADPH oxidase complex. The project has 3 specific objectives, which take advantage of a gp91 phox deficient phagocyte cell line developed by gene targeting as well as heterologous cell systems we have developed for expression of functional recombinant oxidase subunits. First, the relative roles of gp91 phox and p22phox in flavocytochrome b biosynthesis and function will be examined using heterologous cells for expression of unassembled subunits, which are otherwise unstable in phagocytes. Second, identification of domains in gp91 phox and p22 phox that function in the assembly and regulation of oxidase activity will pursued, using a strategy that emphasizes site-directed mutagenesis of candidate hydrophilic regions followed by expression and analysis of function in intact cells. Third, functional domains important for flavocytochrome b trafficking and NADPH oxidase assembly during phagocytosis will be investigated using similar approaches in heterologous and phagocytic cell lines. These studies will provide further insight into the superoxide-generating system of the phagocyte, which may lead to new approaches in modulating superoxide formation in the inflammatory response and host defense.